Construction and expression of TAT-NDPK-A protein and its penetration into the cells

OBJECTIVE To construct the plasmid of pET-TAT-nm23-H1,express the fusion protein of TAT-NDPK-A,and study the penetration of fusion protein into A549 cells.METHODS The fused gene sequences of TAT and nm23-H1 were obtained after PCR with the upstream primer including the DNA sequence of TAT,and then cloned into pET28(a) vector.Recombinant plasmid was sequenced and transformed to Escherichia coli BL21.TAT-NDPK-A was expressed after IPTG induction and purified by Ni2+-NTA affinity column.The antigenicity of fusion protein was analyzed by westen-blot.Fusion protein was added to cultured A549 cells and was observed by fluorescence immunochistochemistry.RESULTS TAT-nm23-H1 was cloned into pET28(a) vector correctly.The purificaton of TAT-NDPK-A protein was 97% after expression induced by IPTG and purification.TAT-NDPK-A protein was delivered into A549 cells by penetrating the cell membrane.CONCLUSION The fusion protein of TAT-NDPK-A is successfully expressed and purified.TAT sequences can deliver the fusion protein to penetrate the cell membrane and enter the cells.It lays solid foundation for the further research on gene function.